Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/067—Hepatocytes
  • C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/11—Epidermal growth factor [EGF]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"

Definitions

• the present invention relates to novel cell surface markers that distinguish hepatic cells from hematopoietic cells, hi particular, the invention relates to methods of isolating bipotent hepatic progenitor cells with a unique phenotype that includes cells that are negative for classical major histocompatibility complex (MHC) class I antigen, positive for the intercellular adhesion molecule 1 (ICAM-l), and dull positive for nonclassical MHC class I antigen(s). Moreover, the invention relates to the hepatic progenitor and hepatic stem cells produced by the methods of the invention.
• MHC major histocompatibility complex
• IAM-l intercellular adhesion molecule 1
• Progenitor cell populations are ideal targets for gene therapy, cell transplantation and for tissue engineering of bioartificial organs (Millar, A.D. 1992 Nature 357, 455; Langer, R. and Vacanti, J.P. 1993 Science 260, 920; Gage, F.H. 1998 Nature 392, 18).
• tissue-specific, "determined” stem cells or progenitors having high growth potential and/or pluripotentiality is readily apparent from studies on hematopoietic stem cells (Spangrude et al. 1988 Science 241, 58), neuronal stem cells (Davis, A.A., and Temple, S. 1994 Nature 372, 263; Stemple, D.L., and Anderson, D.J. 1992 Cell 71, 973) and epidermal stem cells (Jones, P.H., and Watt, F.M. 1993 Cell 73, 713), each having been identified clonally by using the particular methods appropriate for that tissue.
• progenitors are regarded as the cells responsible for normal hematopoietic, neuronal or epidermal tissue homeostasis and for regenerative responses after severe injury (Hall, P.A., and Watt, F.M. 1989 Development 106, 619).
• the mammalian adult liver has a tremendous capacity to recover after either extensive hepatotoxic injury or partial hepatectomy (Fishback, F.C. 1929 Arch. Pathol. 7, 955); (Higgins, G.M., and Anderson, R.M. 1931 Arch. Pathol. 12, 186), even though the liver is usually a quiescent tissue without rapid turnover.
• Data from recent studies in the mouse have been interpreted to suggest that adult parenchymal cells have an almost unlimited growth potentiality as assayed by serial transplantation experiments (Overturf et al. 1997 Am. J. Pathol. 151, 1273); (Rhim, J.A. et al. 1994 Science 263, 1149).
• hepatic cells are intrinsically sensitive to developmental stress stimuli or that the particular microenvironment in fetal liver per se causes such destructive effects (Doi, T.S. et al 1999 Proc. Natl. Acad. Sci. USA 96, 2994).
• the basic architecture of adult liver is dependent on the appearance of the initial cylinder of bile duct epithelium surrounding the portal vein (Shiojiri, N. 1997 Microscopy Res. Tech. 39, 328).
• Immunohistologically, the first sign of the differentiation of intrahepatic bile duct epithelial cells is the expression of biliary-specific cytokeratin (CK).
• CK proteins the cytoplasmic intermediate filament (IF) proteins of epithelial cells
• IF intermediate filament
• CK19 is one of the most remarkable biliary markers, because adult hepatocytes do not express CK19 at all, whereas adult biliary epithelial cells do express this protein.
• Only CK8 and CK18 are expressed through early hepatic cells to adult hepatocytes (Moll, R. et al. 1982 Cell 31, 11).
• MHC major histocompatability
• U.S. Patent No. 5,559,022 to Naughton et al. claims liver reserve cells that bind Eosin Y, a stain that was used to characterize the "reserve cells.”
• U.S. Patent No. 5,559,022 does not use well-established markers for identification of liver reserve cells, nor provide methods for clonal expansion of reserve cells, nor provided markers by which to isolate viable liver reserve cells.
• the present inventors have recognized the inadequacy of growing mature liver cells, such as hepatocytes, rather than the far more useful hepatic progenitors. They have carefully defined the isolation parameters for hepatic progenitors and requirements for clonal growth.
• the progenitor cells and the methods for selecting and culturing the progenitors have many uses, including utility in medicine for treatment of patients with liver failure, and utility for evaluation of toxicity agents, and utility for evaluation of drugs.
• the present invention relates to a method of isolating hepatic bipotent progenitor cells where the cells do not express the classical MHC class I antigen (MHC class la antigen) and do express the ICAM antigen or ICAM-l antigen. Furthermore, the hepatic bipotent progenitor can optionally express nonclassical MHC class I antigen(s) (MHC class lb antigen) containing monomoiphic epitope of MHC class I. Progenitors from several tissues can be used, including, but not limited to, liver. Thus, the invention relates to a method of isolating hepatic progenitor cells that are classical MHC class I negative and, optionally, ICAM-l positive.
• the present invention relates to a method of isolating progenitor cells, where the cells express the phenotype of ICAM-l positive but classical MHC class I negative, by removing cells that express the phenotype classical MHC class I positive.
• the dull expression of nonclassical MHC class I can be used for further isolation of progenitor cells.
• the invention relates to a method of isolating and cloning hepatic pluripotent progenitor cells.
• the hepatic pluripotent progenitor cells may be of any vertebrate species including fish, amphibian, reptilian, avian, and mammalian, and more preferably mammalian.
• the hepatic pluripotent progenitor cells are primate, pig, rat, rabbit, dog, or mouse in origin. Most preferably the pluripotent progenitor cells are human in origin.
• the very most preferable method yields hepatic progenitors that are bipotent hepatic progenitors.
• the bipotent hepatic progenitors can differentiate, or their progeny can differentiate, into either hepatocytes or biliary cells.
• a cell population enriched in progenitors can be obtained by a method of first obtaining a cell suspension of vertebrate cells. Then, sequentially in either order, or substantially simultaneously, the cells that express at least one MHC class la antigen and those that express an IC AM antigen, are removed from the cell suspension to provide a mixture of cells enriched in progenitors. Equally, a mixture of vertebrate embryonic stem cell can be obtained that is enriched in hepatic progenitors by providing a vertebrate embryonic stem cell, expanding the embryonic stem cell to give embryonic stem cell progeny and isolating those embryonic stem cell progeny which express ICAM antigen and do not express MHC class la antigen.
• the methods of separation specifically include the immunoseparations.
• Immunoseparations can be flow cytometry after interaction with a labeled antibody.
• Immunoseparation methods also include affinity methods with antibodies bound to magnetic beads, biodegradable beads, non- biodegradable beads, to panning surfaces including dishes, and to combinations of these methods.
• the hepatic progenitor and bipotent stem cells, and their progeny can optionally express other phenotypes, including, but in no way limited to alpha- fetoprotein, albumin, a higher side scatter than hematopoietic cells from fetal liver, or a pattern of growth as cells that pile up.
• Hepatic stem cells are cells that might or might not express alpha-fetoprotein or albumin but give rise to cells that express alpha-fetoprotein and albumin or biliary markers such as CK19.
• the invention also relates to a method for the identification of progenitor cells, preferably hepatic progenitor cells, by exposing liver cells to a means of detecting a MHC class I phenotype in combination with ICAM-l expression, and identifying those cells within the population that do not express classical MHC class I antigen.
• progenitor cells preferably hepatic progenitor cells
• hepatic progenitor cells by exposing liver cells to a means of detecting a MHC class I phenotype in combination with ICAM-l expression, and identifying those cells within the population that do not express classical MHC class I antigen.
• other markers of progenitor or hepatic phenotypes such as alpha-fetoprotein can be detected.
• the invention additionally relates to hepatic stem and progenitor cells, and their progeny, characterized by a phenotype of classical MHC class I negative and ICAM-l positive, which cells can optionally express other phenotypes, including, but in no way limited to nonclassical MHC class I dull positive, a higher side scatter than hematopoietic cells progenitors, or a pattern of growth as cells that pile up.
• the progeny can express alpha-fetoprotein, albumin, or CK 19.
• the progeny of the hepatic stem and progenitor cells so isolated can retain the parental phenotype and optionally can develop and express additional phenotypes.
• the progeny cells can optionally express the hepatocyte phenotype and the biliary cell phenotype.
• the hepatocyte phenotype is characterized by expression of albumin.
• the biliary cell phenotype is characterized by expression of CK 19.
• composition of hepatic progenitors, their progeny, or a combination of the progenitors and their progeny can also comprise cells that weakly express at least on MHC class lb antigen, exhibit a higher side scatter in flow cytometry than non- parenchymal cells, and express a polypeptide consisting of alpha-fetoprotein, albumin, CK 19, or combinations thereof.
• the composition can be derived from endoderm or bone marrow. In this composition, the endoderm tissue can be liver, pancreas, lung, gut, thyroid, gonad, or combinations thereof.
• Figure 1 A-1C is a characterization of hepatic cell lines from day 15 fetal rat liver.
• Figure 2A-2F is an assay of colony formation on feeder cells.
• Figure 3 A-3X is an expression of rat cell surface antigens on various hepatic cell lines in adult liver cells.
• Figure 4A1-4D4 depicts phenotypic analysis of El 3 fetal rat livers.
• Figure 5A-5D is characterization of hepatic colonies in the absence and presence of EGF.
• Figure 6A-6B depicts induction of CK19 expression on RT1A 1" hepatic cells.
• Figure 7 is a schematic representation of hepatic colony formation on STO5 feeder cells.
• the instant invention is a process for isolation of progenitor cells and a composition comprising progenitor cells.
• the invention is a process for the identification, isolation, and clonal growth of hepatic stem cells and of the hepatic progenitor cells.
• the process involves exposing mixed cell populations derived from an endodermal tissue such as liver to antibodies specific for an ICAM, for example ICAM-l, an adhesion protein, and classical MHC class I antigen, an antigen that characterizes hematopoietic cells and most other nucleated cells but that is substantially absent on the cell surface of hepatic stem cells and progenitors proper.
• the cells can be from any endodermal tissue, including but not limited to liver, pancreas, lung, gut, thyroid, gonad, or from a liver or from a whole organism. Any method of isolating hepatic stem and other early hepatic progenitor cells is acceptable, including by affinity-based interactions, e.g., affinity panning, by immunosurgery in combination with complement or with flow cytometry.
• the flow cytometry separation can also be based on intermediate levels of antigen expression, for example of nonclassical MHC class I antigens.
• the process involves, in addition, selecting for cells that show relatively high side scatter (SSC), a parameter dependent on cellular granularity or amount of cytoplasmic lipid droplets, a feature of hepatic cells.
• SSC side scatter
• the SSC in the hepatic progenitors is higher than in other non- parenchymal cells, such as hematopoietic cells or stromal cells in fetal liver, but lower than in mature parenchymal cells such as those in adult liver.
• other markers expressed on alpha-fetoprotein (AFP)- positive progenitor cells such as CD34, CD38, CD14, and/or CD117, can be used in isolating bipotent progenitor cells.
• AFP alpha-fetoprotein
• markers for the removal of non-hepatic progenitor cells including, but not limited to red blood cell antigen (such as glycophorin A on red blood cells in human liver), immunoglobulin F c receptors, MHC class ⁇ antigens, ABO type markers, CD2, CD3, CD4, CD7, CD8, CD9, GDI la, CDllb, CDl lc, CD15, CD16, CD19, CD20, CD28, CD32, CD36, CD42, CD43, CD45, CD56, CD57, CD61, CD74, CDw75 can be used.
• red blood cell antigen such as glycophorin A on red blood cells in human liver
• immunoglobulin F c receptors such as glycophorin A on red blood cells in human liver
• MHC class ⁇ antigens such as glycophorin A on red blood cells in human liver
• ABO type markers CD2, CD3, CD4, CD7, CD8, CD9, GDI la, CDllb, CDl lc, CD15, CD16, CD19,
• ablative techniques including laser ablation, density separation, sedimentation rate separation including zonal centrifugation, cell elutriation, selective adherence, molecular weighting including cell weighting with tetrazolium salts, size sieving, selective propagation, selective metabolic inhibition including use of cytotoxins, and multi-factor separation.
• the progenitor cells are obtained from a fetus, a child, an adolescent, or an adult.
• hepatic cells be selectively grown in a serum-free, hormone-supplemented, defined medium. It is further preferred that hepatic cells be selectively grown in culture using a layer of feeder cells, where those feeder cells are fibroblasts or another mesodermal cell derivative. It is preferred that the feeder cells are human, non-human primate, pig, rat, or mouse feeder cells, but any mammalian, avian, reptilian, amphibian, or piscine feeder cells are acceptable. It is a yet more preferred embodiment that the feeder cells be embryonic cells, although feeders from neonatal or adult tissue are acceptable.
• the feeder cells be cloned and selected for the ability to support hepatic stem and progenitor cells. It is a still more preferred embodiment of the invention that hepatic stem and progenitor cells be cultured under clonal growth conditions, thereby permitting identification as hepatic cells and expansion of a population of clonal origin.
• One preferred embodiment of the invention comprises mammalian hepatic progenitor cells that are classical MHC class I negative and ICAM-l positive.
• a two color sort is a convenient method to isolate the bipotent cells: ICAM-l positive and classical MHC class I negative are two parameters to define these cells.
• ICAM-l positive cell populations includes hematopoietic, mesenchymal, and mature hepatic cells. The degree of expression is quite variable depending upon the status of the cells (for example, it is different in cells in an activated or quiescent state).
• Classical MHC class I antigen is expressed on all nucleated hematopoietic cells from stem cells to mature cells and on mature hepatocytes (although mature hepatocytes have less expression than hematopoietic cells).
• classical MHC class I negative cells include: bipotent hepatic progenitors, enucleated mature erythrocytes, and an unidentified cell population.
• the cells can express nonclassical MHC class I.
• the progeny of progenitors can express alpha-fetoprotein, albumin, or CK19 and can also exhibit a growth characteristic in which the cells grow in piles on top of each other, that is, in clusters.
• the isolated progenitor cells have the capability to divide and produce progeny. It is further preferred that the progenitor cells are capable of more than about ten mitotic cycles. It is still more preferred that the progeny are progenitor cells or hepatocytes and biliary cells. It is a preferred embodiment of the instant invention that isolated hepatic stem and progenitor cells be
• EGF Epidermal growth factor
• the process involves selecting for cells that additionally express alpha-fetoprotein and bind antibody specific for alpha- fetoprotein. In another preferred embodiment, the process involves selecting for cells that, in addition, synthesize albumin and bind antibody specific for albumin.
• isolated stem and progenitor cells be used as a component of an extracorporeal liver. It is a further more preferred embodiment of the instant invention that the extracorporeal liver having isolated stem and progenitor cells and their progeny be used to support the life of a patient suffering from liver malfunction or failure.
• the invention discloses particular culture conditions that are required for the ex vivo expansion of hepatic progenitor cells, here demonstrated from fetus.
• the inventors selected sublines of STO mouse embryonic cells that proved ideal as feeder cells.
• the feeder cells were used in combination with a novel, serum-free, hormonally defined medium (HDM).
• HDM hormonally defined medium
• the combination enabled the inventors to establish various rat fetal hepatic cell lines from E15 liver in the rat without malignant transformation of the cells.
• the inventor discloses the use of the hepatic cell lines and the HDM-STO co-culture system for development of an in vitro colony forming assay (CFA) for defining clonal growth potential of hepatic progenitors freshly isolated from liver tissue.
• CFA colony forming assay
• the CFA when combined with cells sorted by a defined flow cytometric profile, reveals bipotent hepatic progenitors.
• progenitors from E13 rat livers, corresponding to El 1.5 in the mouse, and with high growth potential have the phenotype as negative for classical MHC class I ( RT1 A region in the rat), dull positive for OX18 (monomorphic epitope on MHC class I antigens), and ICAM-l positive.
• the phenotype of RT1 A negative and OX18 dull positive is equivalent to nonclassical MHC class I (MHC class lb) dull positive.
• EGF is disclosed in this invention to influence both growth of the progenitor colonies and their fates as either hepatocytes or biliary epithelial cells. 6.
• Classical MHC class I antigen The group of major histocompatability antigens commonly found mostly on all nucleated cells although they are most highly expressed on hematopoietic cells.
• the antigen is also known as MDHC class la.
• the nomenclature of the classical MHC antigens is a function of species, for example in humans the MHC antigens are termed HLA.
• Table 3 provides nomenclature of classical MHC antigens in several species.
• Non classical MHC class I antigen The group of major histocompatability antigens, also known as MHC class lb, that can vary even within a species.
• MHC class lb The group of major histocompatability antigens, also known as MHC class lb, that can vary even within a species.
• the nomenclature of the nonclassical MHC antigens varies by species, see, e.g., Table 4.
• ICAM Intercellular adhesion molecule- 1 (CD54) is a membrane glycoprotein and a member of the immunoglobulin superfamily.
• the ligands for ICAM-l are the ⁇ 2-integrin, LFA-1 (CDl la CD18) and Mac-1 (CDl lb/CD18). This molecule is also important for leukocyte attachment to endothelium.
• ICAM-l has a role in leukocyte extravasation.
• ICAM-l is used to designate the form of these molecules found in mammals.
• ICAM or ICAM-l -like are used to designate the homologous and functionally-related proteins in non-mammalian vertebrates.
• Debulking is a process of removing major cell populations from a cell suspension.
• the major non-hepatic lineage cells are red blood cells, macrophages, monocytes, granulocytes, lymphocytes, megakaryocytes, hematopoietic progenitors and stromal cells.
• Dull positive Ln fluorescence-activated cell sorting the intensity of emitted light is proportional to the number of fluorochrome-conjugated immunoglobulin molecules bound to the cell which, in turn, is proportional to the density of the cell surface antigen under study. As the surface density or intracellular density of antigens can vary from a few to hundreds of thousands per cell, a wide range of fluorescence intensities can be measured. The value of dull positive (or dull) is empirically determined and intermediate
• the intensity may also be defined in terms of gates or intensity intervals.
• the dull positive phenotype is a feature of a weakly expressed antigen. The phenotype is also described as weak or low expression.
• Clonal growth In cell culture, clonal growth is the repeated mitosis of one single initial cell to form a clone of cells derived from the one parental cell. The clone of cells can expand to form a colony or cluster of cells. Clonal growth also refers to the conditions necessary to support the viability and mitosis of a single cell. These conditions typically include an enriched and complex basal nutrient medium, an absence of serums, presence of specific growth factors and hormones, substrata of extracellular matrix of defined chemistry, and/or co-cultures of cells that supply one or more of the growth factors, hormones or matrix components.
• the term “remove” means to separate, select and set aside either to retain or discard.
• stromal cells can be removed from a mixed population by any of several means with the intent of either keeping them or of discarding them.
• isolated means to separate from a larger group and keep apart.
• progenitor cells can be isolated from a mixed population of progenitor and non-progenitor cells.
• purify means to separate away unwanted components.
• Hepatic progenitor cells frequently exhibit a distinctive feature, in which the cells divide and remain in mutual proximity.
• the progenitor cells form clusters in which cells are piled up one on another. Cells in the three- dimensional mass of piled-up cells are adjacent to feeder cells or to other progenitor cells.
• the clusters are also termed P-colonies or P-type colonies and are distinct from cell monolayers.
• Pregnant Fisher 344 rats are obtained from Charles River Breeding Laboratory (Wilmington, MA). For timed pregnancies, animals are put together in the afternoon, and the morning on which the plug is observed is designated day 0. Male Fisher 344 rats (200-250g) are used for adult liver cells.
• hepatic cell lines from embryonic day 15 livers. Fetal livers are prepared from day 15 of the gestation. Single cell suspensions are obtained by incubating the livers with 0.05% trypsin and 0.5mM EDTA or lOunits/ml thermolysin (Sigma, St. Louis, MO) and lOOunits/ml deoxyribonuclease I (Sigma) for at 37°C. The cells are overlayed on Ficoll-paque (Pharmacia Biotech, Uppsala, Sweden) for gradient density centrifugation at 450g for 15 min.
• Ficoll-paque Pulcoll-paque
• the cells from the bottom fraction are inoculated into tissue culture dishes coated with 17 mg/ml collagen type IV (Collaborative Biomedical Products, Bedford, MA) or 12 ⁇ g/ml laminin (Collaborative Biomedical Products) for thl 120-3 and rter ⁇ or rhel4321, respectively.
• the serum-free hormonally defined culture medium, HDM is a 1 : 1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 (DMEM/F12, GIBCO/BRL, Grand Island, NY), to which is added 20 ng/ml EGF (Collaborative Biomedical
• trypsinized cells are cultured on a feeder layer of mitomycin C- treated STO mouse embryonic fibroblast line (American Type Culture Collection, Rockville MD).
• Thl 120-3, rter6, and rhel4321 are cloned from three independent preparations of fetal hepatic cells and are maintained on STO feeder cells with HDM.
• the concentration of EGF is reduced to 10 ng/ml for all cell cultures.
• Fetal livers are dissected into ice-cold Ca ++ free HBSS with lOmM HEPES, 0.8mM MgSO4 and ImM EGTA (pH7.4).
• the livers are triturated with 0.2% type IV collagenase (Sigma) and 16.5 units/ml thermolysin (Sigma) in HBSS prepared with lOmM HEPES, 0.8mM MgSO4, and ImM CaCl2- After incubation at 37°C for 10 min, the cell suspension is digested with 0.025% trypsin and 2.5mM EDTA (Sigma) for 10 min.
• Trypsin is then quenched by addition of lmg/ml trypsin inhibitor (Sigma). Finally, the cells are treated with 200 units/ml deoxyribonuclease I (Sigma), all experiments, 3-5 x lO ⁇ cells per liver are obtained.
• liver perfusion method Isolation of adult liver cells.
• the two step liver perfusion method is performed to isolate liver cells. After perfusion, the cells are centrifuged for 1 min at 50g twice to enrich for large parenchymal cells. Cellular viability is >90% as measured by trypan blue exclusion.
• STO Sublines One hundred cells of parent STO from ATCC are cultured in 100mm culture dishes for 7 days in DMEM/F12 supplemented with 10% heat-inactivated fetal bovine serum, 2 x lO ⁇ M glutamine, 5 x lO ' ⁇ M 2-mercaptoethanol and antibiotics. Four subclones are selected for further characterization according to the cell morphology and the growth speed. Although CFA for rter6 is performed in the four subclones, one of them, STO6, does not persist in attaching to culture plates after mitomycin C-treatment. One subclone, STO5, is transfected with pEF-Hlx-MClneo or pEF-MClneo kindly provided from Dr. J. M. Adams, The Walter and Eliza Hall
• anti-CK19 monoclonal antibody (Amersham, Buckinghamshire, England) and FITC-conjugated anti mouse IgG (Caltag, Burlingame, CA) are used instead of anti alpha-fetoprotein antibody.
• the established three hepatic cell lines are trypsinized and fractionated by Percoll density gradient centrifugation to remove feeder cells.
• the rat hepatoma cell line, FTO-2B, and the rat liver epithelial cell line, WB-F344, as well as adult liver cells are stained to compare with the fetal hepatic cell lines.
• the cell lines are kind gifts of Dr. R.E.K. Fournier, Fred Hutchinson Cancer Research Center, Seattle, WA, and Dr. M.-S. Tsao, University of North Carolina, Chapel Hill, NC, respectively. Cells are blocked and stained with FITC-
• FITC-conjugated anti mouse IgG is used for OX18.
• Cell suspensions of three fetal hepatic cell lines are stained with biotinylated anti- mouse CD98 followed by a second staining with streptavidin-red670 as well as anti- rat moAb to gate out mouse cell populations.
• CFA for hepatic cell lines, sorted cells, and adult liver cells.
• the hepatic cell lines are plated in triplicate at 500 cells per 9.6 cm ⁇ on mitomycin C-treated STO feeder layer with the same HDM as used for maintaining each cell line. Before plating, cell are trypsinized and fractionated by Percoll density gradient centrifugation to remove feeder cells. The cultures are incubated for 10 to 14 days with medium changes every other day. Double immunofluorescence staining of alpha-fetoprotein and albumin is then performed. 100 colonies per well are analyzed by the colony morphology, P or F type, and the expression of alpha-fetoprotein and albumin.
• the colonies are stained using Diff-Quick (Baxter, McGaw Park, IL) to count the number of the colonies per well.
• Diff-Quick Biller, McGaw Park, IL
• the plating cell number is changed as described.
• the culture period is expanded to between 14 and 17 days, and the concentration of dexamethasone is increased to 10 ⁇ 6M. All other procedures are performed as above.
• small numbers of clumps of liver cells are not eliminated from the cell suspension after the preparation. Therefore, an undefined number of the colonies might be produced from the clumps.
• double immunofluorescence staining of albumin and CK19 of the colonies is performed at 5 days each of the culture in the presence or absence of EGF.
• any colony with more than one CK19 + cell is counted as a CK19+ colony.
• colonies containing multiple clusters of two CK19 + cells or one cluster of more than three CK19+ cells are counted as a CK19+ colony.
• About 100 colonies per well are counted. Each point represents the mean ⁇ SD from triplicate-stained cultures.
• hepatic cell lines from independent experiments are selected by the morphological criteria of either P type or F type colonies (Fig. 1 A-1C).
• Rhel4321 (Fig. 1A) consists mostly of packed small cells, P type colonies, whereas thl 120-3 (Fig 1C) makes only a flattened monolayer of F-type colonies.
• Rter6 (Fig. IB) is an intermediate phenotype of these two. Interestingly, the heterogeneity of rter6 is still observed after three rounds of sequential cloning of the flattened colony.
• Fig. 2A-2F shows the
• the total colony number per well is counted to calculate the clonal growth efficiency (colony efficiency).
• the efficiency of rter6 and rhel4321 is 45.7% ( ⁇ 1.3% SD) and 36.4% ( ⁇ 1.1% SD), respectively.
• the thll20-3 cells tightly attach to each other along their lateral borders making preparation of single cell suspensions difficult. However, the thl 120-3 cells do not produce piled up clusters (Fig. 1C).
• the culture system To develop a CFA system to identify bipotent hepatic progenitors with high growth potential, the culture system has to be able to support cell expansion at clonal seeding densities and with conservation of critical original hepatic functions. Albumin and alpha-fetoprotein are two of the most significant markers for early hepatic development. The culture conditions optimizing P type colonies should be the best, since
• STO subclones are compared in their support of P type colonies of rter6.
• One of the clones, STO5 supports the P type colony formation more than any of the other sublines and more than the parent line (Fig. 2D).
• the CFA of rhel4321 also confirms that STO5 is a more effective feeder than the parent STO (Fig. 2E).
• the mouse Hlx gene product expressed in the mesenchymal cells lining digestive tract from E10.5, is essential for fetal hepatic cell expansion.
• Hepatopoiesis and massive amounts of hematopoiesis co-exist in the fetal liver. So far, the antigenic profile of hematopoietic progenitors has extensively been analyzed, whereas studies of early hepatic progenitors are still in their infancy. The antigenic profile of hepatic cells is analyzed using the three hepatic cell lines established in this study, an adult hepatocarcinoma cell line (FTO-2B), an epithelial cell line from adult rat liver (WB-F344), and freshly isolated adult liver cells (Fig. 3A-3X).
• FTO-2B adult hepatocarcinoma cell line
• WB-F344 epithelial cell line from adult rat liver
• Fig. 3A-3X freshly isolated adult liver cells
• rhel4321 Compared with FTO-2B, WB-F344, and adult liver cells, the pattern of the most immature of the fetal hepatic cell lines, rhel4321, is quite unique in that there is no expression of classical MHC class I (RT1A 1 ) (Fig. 3 A).
• the cell line thl 120-3 is similar to rhel4321 in the pattern of RT1A 1 (Fig. 31), OX18 (pan-MHC class I) (Fig. 3J), and ICAM-l (Fig. 3K), whereas rter6 has relatively high expression of RT1A 1 (Fig. 3E) and OX18 (Fig. 3F).
• RT1A 1+ (Fig. 3U), OX18 + (Fig. 3V), and ICAM-1 + (Fig. 3W). Since, in the adult rat, all bone marrow cells except mature erythrocytes strongly express MHC class I molecules, the fetal hepatic population can be separated from the hemopoietic cell populations by MHC class I expression. The cell suspensions from rat E13 livers are stained with anti RT1A 1 and ICAM-l antibodies. Fig. 4A1 to 4A2 shows the
• Fig. 4B-1 to 4B-5 represent the result of resorting of the five fractions after sorting.
• the hepatic cell colonies defined by expression of albumin and alpha- fetoprotein, are distinguishable also morphologically, enabling one to count the number of hepatic colonies per well. The majority of the hepatic colonies are detected in the gate RTlA lduI1 and ICAM- 1" (Table 1, Fig. 4B-2, i.e.
• gate 2 shows a much lower number of the colonies, and the other fractions contain negligible numbers of cells with colony forming ability.
• gate 1 shows a much lower number of the colonies, and the other fractions contain negligible numbers of cells with colony forming ability.
• the expression of both alpha-fetoprotein and albumin is confirmed in all the hepatic colonies. Some of the colonies, derived from cells in gate 2, are larger than others.
• SSC sidescatter
• SSC Sidescatter
• EGF has long been known as a potent growth factor for adult liver cells. Therefore, the effects of EGF for colony formation of sorted hepatic cells are investigated.
• the colony-size of the RT1A 1_ OX18 du11 , ICAM-1 + hepatic cells becomes bigger in the absence of EGF, whereas adult liver cells yielded colonies only in the presence of EGF (Fig. 5A).
• the morphology of the colonies derived from adult liver cells is the typical F type, whereas all RT1A 1" hepatic cells produce P type colonies without EGF.
• the expression of RTIA 1" , OX18, and ICAM-l is assessed. As shown in Fig. 5B to 5D, the expression of RTIA 1 is not induced, while that of OX18 is reduced. The level of ICAM-l does not change. Furthermore, the average cell number of single colony is calculated from the recovered cell number, the percentage of rat hepatic cells and the colony efficiency. The estimated cell number reaches 3 to 4 x 10 3 (Table 2). This indicates that the single cell forming the colonies divided approximately 11-12 times on average under this culture condition.
• Sorted cells from R4 in Fig. 4C-5 were cultured on STO5hlx feeder cells in 60mm or 100mm dish. After the period indicated of the culture cell all cells were recovered and the toal cell number counted. The percentage of rat cells is from flow cytometric analysis based on the expression of rat ICAM-l and mouse CD98. Colony efficiency indicates the percentage of cells inoculated to culture that went on to form colonies. Data from triplicate-stained cultures (mean) was obtained from the experiments run parallel with.
• CK19 is expressed in the bile duct epithelial precursors after day 15.5 in the fetal rat liver at which time the expression of albumin disappears in the cells.
• the sorted RTIA 1" ICAM-1 + cells are cultured in the presence or absence of EGF, and their fates are monitored by the expression of CK19 and albumin after 5 days of culture. After the first 5 days, the CK19 + colonies are negligible in the cultures treated with EGF, whereas a few colonies containing CK19 + cells occurred in those in the absence of EGF (Fig. 6a to 6b). Although the intensity of the CK19 expression is fairly weak, the CK19 + cells show reduced albumin expression. At the 10th day of the culture, as shown in Fig.
• MHC antigens are highly conserved among vertebrates, and the same is the case for the ICAM antigens.
• MHC antigens are not found in invertebrates. MHC antigens are the most comprehensively investigated molecules of vertebrate species. Although the information on ICAM antigens is limited, the biological functions of ICAM antigens are conserved in many mammals such as human mouse, and rat. So far, ICAM-l complementary DNA has been cloned from human, chimpanzee, mouse, rat, dog, and bovine. The conclusion from the sequence data is that the molecular structure is highly conserved in all species. Therefore, by choosing antibodies specific for the ICAM-l in a given species and antibodies for the designated class I MHC antigen according to the table, the cell populations enriched in hepatic progenitor cells can be isolated.
• OX18 recognizes a monomorphic epitope of rat MHC class I antigens. Therefore, the antibody recognizes nonclassical MHC class I as well as classical MHC class I.
• the exact number of nonclassical MHC class I loci are not defined in any species, because it varies between members of the same species. Therefore, in the future, a new locus might be discovered as a nonclassical MHC class I in subpopulations of these species.
• One embodiment of the invention is a method of predicting the phenotype of hepatic progenitor cells. This feature is illustrated in the table of key cell surface markers in various species.
• MHC class I 2D negative and/or H-2L negative negative and/or HLA- B negative and/or HLA- C negative
• EGF epidermal growth factor that when added to the culture conditions appears to drive the cells towards the hepatocytic lineage and blocks development of the biliary lineage, h the absence of EGF, there is spontaneous differentiation towards both biliary and hepatocytic lineages.
• CK 19 is not expressed on adult hepatocytes in vivo. However, in any culture of adult liver cells, one can observe one or two cells that express some CK19 but without apparent inducibility by culture conditions and without distinctions morphological
• ANTIGENIC PHENOTYPING OF HUMAN FETAL LIVER CELLS Human fetal liver cells are stained with antibody to CD 14.
• CD 14 Several populations are identified by two-color cell sorting of HLA (ABC) vs. CD14. These populations include a group designated R2 characterized by intermediate HLA staining and without CD 14 staining and another group designated R3 characterized by high CD 14 staining and high HLA staining. When stained for alpha-feto protein, the R3 cells are positive for alpha-fetoprotein and the R2 contains two subpopulations, only one of which stains for AFP.
• the cell suspension is incubated with fluorescein-conjugated antibody to the HLA class I monomorphic epitopes.
• fluorescein-conjugated antibody to the HLA class I monomorphic epitopes.
• any of many other fluoro chromes can be used in place of fluorescein, including, but not limited to rhodamine and Texas Red.
• indirect-immuno fluorescence is used to label the cells. That is, the fluorescent label is conjugated to an antibody directed to the immunoglobulin of the species in which the primary antibody is elicited.
• the cell sample is sorted by high throughput fluorescence - activated cell sorted using any of a variety of commercially available or customized cell sorter instruments. Hepatic progenitor cells that have intermediate or dull fluorescence with the labeled anti-monomorphic epitopes are selected.
• Compositions enriched in rat hepatic progenitors can also be advantageously prepared by sorting liver cell suspensions using antibodies to CD44H.
• Liver cells that show a high level of sidescatter also express CD44H and express alpha fetoprotein.
• cells that express alpha-fetoprotein also express higher levels of CD44H.
• liver cells that have a low level of sidescatter do not express CD44 at higher levels.
• antibodies specific for polymorphic epitopes including but not limited to, HLA-A2, HLA-B27, and HLA-Bw22, are used to identify and isolate hepatic progenitors.
• antibodies specific for nonclassical HLA class I antigens including HLA-G, HLA-E, and HLA-F, are used to identify and isolate hepatic progenitor cell that express the antigen.
• red blood cells component can be advantageously used and these methods can reduce some of the stromal cell population as well.
• These methods include fractionation on Percoll gradients and specific depletion using antibody to glycophorin A, CD45, or both.
• these methods include sedimentation velocity, separation in density gradients other than Percoll, e.g., Ficoll, zonal centrifugation and cell elutriation.
• Percoll e.g., Ficoll
• zonal centrifugation e.g., zonal centrifugation and cell elutriation.
• Isolation of cell populations that are positive for ICAM-l and negative for classical MHC class I antigen are further characterized with other markers including nonclassical MHC class I to identify hepatic progenitors.
• progeny of these progenitor cells labeled with antibodies to the cytoplasmic proteins, such as alpha-fetoprotein and/or albumin markers that are long-known to be characteristic of hepatic progenitors.
• Alpha-fetoprotein and albumin are representative of the well known markers for hepatic progenitors that cannot be used to select for viable cells, since labeling the cells for those proteins requires permeabilization of the cells, a process that destroys their viability.
• cell samples from a population can be tested for alpha-fetoprotein, albumin, and cytokeratin. Thereby, the characteristics of the whole population are deduced.
• the high correlation between the cell surface markers e.g., ICAM-l positive, OX-18 dull positive, classical MHC class I negative
• clonal growth capability demonstrates that viable cells can be isolated using selection for the surface markers alone.
• hepatic progenitors are highly granular (show very high side scatter); the hepatic progenitors are intermediate in granularity; and the non-parenchymal cell populations have even less granularity than the hepatic precursors.
• the hepatic progenitors In cells from fetal tissue, consisting almost entirely of non-parenchymal cells and hepatic progenitors, the hepatic progenitors have the highest granularity.
• Hepatic progenitors are selected as the cell population that is intermediate in granularity by flow cytometry.
• Compositions enriched in human hepatic progenitors can also be advantageously prepared by sorting liver cell suspensions using antibodies to CD 14 in combination with antibodies to HLA, the human version of MHC.
• R2 which express relatively intermediate levels of HLA and do not express CD 14
• R3 which express relatively high levels of HLA and relatively high levels of CD 14.
• the R2 cells are further characterized to have two subpopulations by expression of alpha-fetoprotein.
• the R3 cells are further characterized to consist only of cells that express alpha fetoprotein.
• the hepatic progenitors are distinguished from red blood cells by use of monoclonal antibodies (Glycophorin A for human) and a polyclonal antiserum to red blood cell antigen if monoclonal antibodies are not available. Also, cells that express common leukocyte antigen (CD45) also express classical MHC class I antigen. Therefore, by default, CD45 is not an antigen that can be used to identify the rodent
• hepatic progenitors including nonclassical HLA class I antigens, ICAM-l and alpha-fetoprotein can be used to characterize liver cancers to better define successful treatments of those cancers.
• Cancers in general, are transformants of stem cells and early progenitor cell populations. However, these transformants often retain expression of the antigenic markers shared with their normal counterparts. Liver cancers, distinguished by these antigenic markers, can identify cancers responding in distinct ways to oncological therapeutic modalities (e.g., chemotherapeutic drugs, radiation, and adjuvant therapies).
• ES cells embryonic stem cells
• ES cells are becoming popular as possible all-purpose stem cells for use in reconstitution of any tissue.
• past studies of injection of ES cells into tissues resulted in tumors, some of which were malignant.
• the only way the ES cells are to be used clinically is to differentiate them to determined stem cells and then inject the determined stem cells.
• embryonic stem cells are maintained in cell culture under culture conditions that permit proliferation to form progeny.
• the ES progeny are subjected to flow cytometry after incubation with antibodies to classic MHC class I and ICAM-l antigens.
• ES progeny meeting the criteria for hepatic progenitors are expanded in cell culture.
• the markers we have identified can be used to define an hepatic fate for a determined stem cell.
• liver progenitor cells are used to identify cell populations for gene therapies. To date, gene therapies have often not worked or not worked well with targeting to mature cell populations. The major successes in gene
• ex vivo gene therapies in hemopoietic progenitor cell populations. Therefore, ex vivo gene therapies for liver are used with hepatic- determined stem and progenitor cells isolated by our protocols. Also, the gene therapies involving "targeted injectable vectors" are improved by focusing on those that target hepatic progenitors.
• Hepatic progenitors can be used for gene therapy as follows:
• Phenylketonuria is an autosomal recessive disorder caused by a deficiency of phenylalanine hydroxylase (PAH) in the liver.
• PAH phenylalanine hydroxylase
• Patients with PKU show profound mental retardation and hypopigmentation of skin, hair, and eyes due to increased amount of phenylalanine in body fluids.
• the rigid dietary restriction significantly reduces serum phenylalanine levels, reduced compliance, even in adolescence or early adulthood, often leads to a decline in mental or behavioral performance.
• Gene therapy technique is one alternative to dietary therapy for PKU.
• hepatic progenitors with high growth potentiality can eliminate the problem mentioned above.
• liver failure is modeled by surgical removal of about 70% of the liver and ligation of the common bile duct in an experimental group of ten male rats (125 to 160 g body weight).
• a sham control group of ten age- and sex-matched rats is subjected to s similar anesthesia, mid-line laparotomy, and manipulation of the liver, but without ligation of the bile ducts and without hepatectomy.
• livers of 12 embryonic (embryonic day 14) rat pups are aseptically removed, diced, rinsed in ImM EDTA in Hank's BSS without calcium or magnesium, pH 7.0, then incubated for up to 20 minutes in Hank's BSS containing 0.5 mg/ml collagenase to produce a near single cell suspension.
• Bipotent hepatic progenitors are prepared by any of the above methods.
• the rats both experimental and sham control, are subjected to a 5 mm abdominal incision to expose the spleen.
• the number of cells administered to different groups of animals can be about 10 3 up to about 10 10 , in particular, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 s , 10 9 and 10 10 .
• the immunosuppressant cyclosporine A 1 mg/kg body weight, is administered daily intraperitoneally.
• Blood levels of bilirubin, gamma glutamyl transf erase and alanine aminotransferase activities are monitored two days before the hepatectomy or sham hepatectomy operation and on post-operation days 3, 7, 14, and 28. Body weight, water consumption, and a visual inspection of lethargy are recorded on the same days. At 28 days post hepatectomy all surviving animals are killed for histological evaluation of spleen and liver.

Landscapes

• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Chemical & Material Sciences (AREA)
• Biomedical Technology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Biotechnology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Animal Behavior & Ethology (AREA)
• Medicinal Chemistry (AREA)
• Zoology (AREA)
• Veterinary Medicine (AREA)
• Gastroenterology & Hepatology (AREA)
• Wood Science & Technology (AREA)
• Genetics & Genomics (AREA)
• Obesity (AREA)
• Cell Biology (AREA)
• Diabetes (AREA)
• Microbiology (AREA)
• Developmental Biology & Embryology (AREA)
• Hematology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (1)

Publications (2)

Family

ID=21741847

Family Applications (1)

Country Status (11)

Families Citing this family (12)

Family Cites Families (2)

• 2000
  • 2000-10-03 CA CA2424781A patent/CA2424781C/fr not_active Expired - Fee Related
  • 2000-10-03 ES ES00968708T patent/ES2367005T3/es not_active Expired - Lifetime
  • 2000-10-03 AT AT00968708T patent/ATE510905T1/de active
  • 2000-10-03 AU AU7858200A patent/AU7858200A/xx active Pending
  • 2000-10-03 IL IL15524900A patent/IL155249A0/xx unknown
  • 2000-10-03 AU AU2000278582A patent/AU2000278582B2/en not_active Ceased
  • 2000-10-03 DK DK00968708.8T patent/DK1325112T3/da active
  • 2000-10-03 JP JP2002532568A patent/JP2004510432A/ja active Pending
  • 2000-10-03 CN CNB008200483A patent/CN1258589C/zh not_active Expired - Fee Related
  • 2000-10-03 WO PCT/US2000/027429 patent/WO2002028997A2/fr active Application Filing
  • 2000-10-03 EP EP00968708A patent/EP1325112B1/fr not_active Expired - Lifetime
• 2003
  • 2003-04-03 IL IL155249A patent/IL155249A/en not_active IP Right Cessation
  • 2003-09-19 HK HK03106717.6A patent/HK1054569B/zh not_active IP Right Cessation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events